The androgen receptor (AR) plays an important role in the development and progression of prostate cancer (PCa). Most PCa specimens express high levels of AR, which is necessary and sufficient to convert androgen-dependent (AR+) PCa to the clinically more aggressive androgen-independent (AR-) PCa. Although inhibition of AR activity is a mainstay of AR+ PCa treatment, there are no effective therapies for AR- PCa, which is uniformly lethal. Studies reveal AR and Akt mutually activate one another, and that together AR and Akt inhibit pro-apoptotic signaling and enhance the conversion of AR+ to AR- PCa. Targeting both AR and Akt signaling represents a powerful therapeutic approach for treating AR+ PCa and preventing the emergence of AR- PCa. We recently demonstrated Withaferin-A (WA), a natural compound, specifically targets AR- PCa cells by inhibiting phosphorylated Akt; this inhibition leads to the activation of prostate apoptosis response-4 (Par-4)-dependent apoptosis in cell culture and in animal models. Similarly, inhibition of AR in AR+ PCa cells facilitates WA-induced Par-4 activation and apoptosis. Over-expression of either Akt or AR inhibits WA-mediated Par-4 activation, and blocks apoptosis in AR+ PCa, suggesting both AR and Akt negatively regulate the pro-apoptotic functions of Par-4 in PCa cells. We hypothesize that WA in combination with hormone ablation is an effective strategy to control the progression of PCa. To address this hypothesis, we will: Aim 1. Characterize the molecular link between AR signaling and Par-4, and determine the effects of WA on AR+ PCa cells (investigating AR binding to the Par-4 promoter via reporter constructs, ChIP assays using AR antibodies, and Western analysis of pro- and anti-apoptosis proteins); Aim 2. Examine the relationship between Par-4 and FOXO 3a, which is regulated by Akt, and how WA modulates this interaction in AR+ PCa (studying FOXO3a binding to the Par-4 promoter via reporter constructs, ChIP assays using FOXO3a antibodies, mobility shift assays, Western analysis of pro- and -anti-apoptosis proteins, and TUNEL and annexin-V assays following FOXO3a over-expression); and Aim 3. Determine the chemotherapeutic effects of WA on the development of PCa in prostate specific PTEN knockout mice (PS PTEN-KO; performing in vivo WA efficacy studies on PCa, histopathology, immunohistochemistry of pro-survival and pro-apoptotic proteins, proliferation index, apoptosis, and serum WA levels). We anticipate these studies will provide molecular insight into the mechanism of WA action against AR+ PCa, and will ultimately lead to novel therapeutic strategies for the treatment of PCa.